Pollen-mediated transfer of herbicide resistance between johnsongrass (Sorghum halepense) biotypes

Johnsongrass (Sorghum halepense) is a troublesome weed in row crop production in the United States. Herbicide resistance is a growing concern in this species, with resistance to ACCase-, ALS-, and EPSPS-inhibitors already reported. Pollen-mediated gene flow (PMGF) is capable of spreading herbicide resistance, but the extent of PMGF has not yet been studied in johnsongrass. Field experiments were conducted in a Nelder-wheel design to quantify the distance and frequency of PMGF from ALS-inhibitor-resistant (AR) to -susceptible (AS) johnsongrass across three environments (summer 2018, fall 2018, and fall 2019). The AR biotype (pollen donor) was established at the center of the wheel (5-m diameter), and a naturally occurring johnsongrass (AS) infestation was utilized as the pollen recipient, in eight directions and at nine distances (5, 10, 15, 20, 25, 35, 40, 45, and 50 m) within each direction. Seeds collected from the AS plants in each distance and direction were screened for survival to the ALS-inhibitor herbicide nicosulfuron (Accent Q) at 95 g ai ha−1 under greenhouse conditions. The survivors (i.e. hybrids) were further confirmed based on the presence of the Trp574Leu mutation. At the closest distance of 5 m, PMGF was 9.6–16.2% across the directions and environments, which progressively declined to 0.8–1.2% at 50 m. The exponential decay model predicted 50% reduction in PMGF at 2.2 m and 90% reduction at 5.8 m from the pollen donor block. Results demonstrate that herbicide resistance can spread between adjacent field populations of johnsongrass through PMGF, which necessitates sound monitoring and management.

www.nature.com/scientificreports/ pot, 5 seeds were planted and thinned to one healthy seedling at 1-leaf stage. Seedlings were supplied with sufficient water and nutrients (Miracle-Gro Water Soluble All Purpose Plant Food, Scotts Miracle-Gro Products Inc., 14111 Scottslawn Road, Marysville, OH 43041). A total of 50 seedlings were established in the greenhouse and were maintained until they reached about 10 cm tall, at which point they were sprayed with 2× the field rate of nicosulfuron (63 × 2 = 126 g ai ha −1 ) (mixed with 0.25% v/v COC). The herbicide was applied using a tracksprayer (Research Track Sprayer, DeVries, Hollandale, MN) fitted with a flat fan nozzle (TeeJet XR110015) that was calibrated to deliver a spray volume of 140 L ha −1 at 276 kPa pressure, and at an operating speed of 4.8 kmph.
All treated seedlings that survived the herbicide application at 21 days after treatment (DAT) were then used as the pollen donor in the field gene flow experiment. All plant materials were handled in accordance with relevant guidelines and regulations. No permissions or licenses were required for collecting the johnsongrass samples from the experimental fields.
Dose-response assays. The degree of resistance/susceptibility to nicosulfuron of the AR and AS johnsongrass biotypes were determined using a classical dose-response experiment. The assay consisted of seven rates (0, 0.0625, 0.125, 0.25, 0.5, 1, and 2×) for the AS population and nine rates (0, 0.25, 0.5, 1, 2, 4, 8, 16, and 32×) for the AR population [1 × (field recommended rate) = 63 g ai ha −1 of Accent Q]. The experimental units were arranged in a completely randomized design with four replications. Seeds of AR and AS plants were planted in plastic trays (25 × 25 cm) filled with commercial potting-soil mix (LC1 Potting Mix, Sungro Horticulture Inc., Agawam, MA, USA) and maintained at 26/22 °C day/night cycle with a 14-h photoperiod in the greenhouse. Seedlings at 1-2 leaf stage were thinned to 20 seedlings per tray; four replications each of 20 seedlings per dose were considered. The seedlings were watered and fertilized as needed. The assay was conducted twice, thus a total of 160 seedlings were screened for each dose. The established seedlings were sprayed with the appropriate herbicide dose at the 10-15 cm tall seedling stage. The herbicide was applied using a track sprayer calibrated to deliver a spray volume of 140 L ha −1 at 4.8 kmph operating speed. Survival (%) and injury (%) were assessed at 28 DAT. Any plant that failed to grow out of the herbicide impact was considered dead. Plant injury was rated for each plot (i.e. on the 20 seedlings per rep) on a scale of 0-100%, where 0 indicates no visible impact compared to the nontreated control and 100 indicates complete death of all plants in the tray. Immediately after the visual ratings were completed, shoot biomass produced by the 20 plants from each tray was determined by harvesting all the tissues at the soil level and drying them in an oven at 60 °C for 72 h. Seedling mortality data were used for fitting dose-response curves that allowed for determining the lethal dose that caused 100% mortality of the susceptible biotype. This dose was used as a discriminant dose to distinguish between a hybrid (that confers resistance to nicosulfuron as a result of gene flow) and a selfed progeny (susceptible to nicosulfuron) in the field gene flow study.
Field experimental location and set-up. The field experiment was conducted across two ENVs in 2018 (summer and fall) and one in 2019 (fall) at the Texas A&M University Farm, Somerville (Burleson County), Texas (30° 32′ 15.4″ N 96° 25′ 49.2″ W). The study site is characterized by silty clay loam soil with an average annual rainfall of 98.2 cm. The field experiment followed the Nelder-wheel design 40 , i.e. concentric donorreceptor design, a widely used method for gene flow studies, wherein the pollen-donors are surrounded by the pollen-receptors (Fig. 1). In this study, the AR plants (planted in the central block of the wheel) served as the pollen-donors, whereas the AS plants (present in the spokes) served as the pollen-receptors.
The center of the wheel was 5 m in diameter, and each spoke was 50 m long starting at the periphery of the central circular block. Thirty AR plants (pollen-donors) were transplanted in four concentric rings of 1, 5, 9, and 15 plants in the 5 m diameter central block, surrounded by the pollen-receptors (i.e. AS plants) (Fig. 1). The AR plants were contained within the central block during the 2 years of the field experiment by harvesting and removing all mature seeds and removing any expanding rhizomatous shoots. Further, field cultivation was completely avoided in the central block throughout the study period. Any newly emerging johnsongrass plants (seedling/rhizomatous) other than the transplanted AR plants in the central block were removed periodically by manual uprooting.
The wheel consisted of eight spokes (i.e. directional blocks) arranged in four cardinal (N, E, S, W) and four ordinal (NE, SE, NW, SW) directions (Fig. 1). The plots to quantify gene flow frequency were arranged at 0 (border of the central block), 5,10,15,20,25,30,35,40,45, and 50 m distances from the central block in all eight directions (Fig. 1). Each plot measured 3 × 2 m and the area surrounding the plots was shredded prior to the booting stage with a Rhino ® RC flail shredder (RHINOAG, INC., Gibson City, IL 60936).
A tall-growing biomass sorghum border (6 m wide) was established surrounding the experimental area in all directions to prevent potential inflow of pollen from other Sorghum spp. in the nearby areas. Additionally, prevailing weather conditions, specifically wind direction, wind speed, relative humidity, and air temperature measured at 5-min intervals were obtained from a nearby weather station located within the Texas A&M research farm (http:// afs102. tamu. edu/). The field did not require any specific agronomic management in terms of irrigation, fertilization, or pest management.
Flowering synchrony, tagging, and seed harvesting. At peak flowering, when > 50% of the plants in the AR block started anther dehiscence (i.e., pollen shedding), ten AS panicles (five random plants × 2 panicles per plant) that showed flowering synchrony with AR plants and displayed protruded, receptive stigma were tagged using colored ribbons at each distance and direction. At seed maturity, the tagged AS panicles were harvested separately for each distance and direction. Panicles were threshed, seeds were cleaned manually, and stored under room conditions until used in the herbicide resistance screening to facilitate after-ripening and dormancy release. To effectively detect the levels of gene flow from AR to AS biotypes especially at low frequencies, the minimum sample size required for resistance screening was determined based on the following formula (Eq. 1) 41 : where P is the probability of detecting a resistant progeny in the least frequent class and p is the probability of the least frequent class. Based on this formula, a minimum of 298 to as high as 916 plants were screened for each distance within each direction, allowing for a 1% detection level (p = 0.01) with a 95% (P = 0.95) confidence interval.
Approximately one-year old progeny seeds harvested from the AS plants were scarified using a sandpaper for 15-20 s to release dormancy. The seeds for each distance within each direction were planted in four replicates of plastic trays (50 × 25 cm) filled with potting soil mixture (LC1 Potting Mix, Sungro Horticulture Inc., Agawam, MA, USA). The plants were raised at the Norman Borlaug Center for Southern Crop Improvement Greenhouse Research Facility at Texas A&M University. The greenhouse was maintained at 28/24 °C day/night temperature regime and a 14-h photoperiod. About 10-15 cm tall seedlings were sprayed with the discriminant dose of the ALS-inhibitor nicosulfuron (Accent Q, 95 g ai ha −1 ) using a spray chamber (Research Track Sprayer, DeVries, Hollandale, MN) fitted with a flat fan nozzle (TeeJet XR110015) that was calibrated to deliver a spray volume of 140 L ha −1 at 276 kPa pressure, operating at a speed of 4.8 kmph. At 28 DAT, percent seedling survival was determined based on the number of plants that survived the herbicide application out of the total number of plants sprayed. The number of plants in each tray was counted before spraying.

Molecular confirmation of hybrids.
Leaf tissue samples were collected from thirty random surviving plants (putative resistant) in the herbicide resistance screening study for each of the three field ENVs, thus totaling 90 samples. Genomic DNA was extracted from 100 mg of young leaf tissue using the modified CTAB protocol 42 . The concentration of DNA was determined using a Nanodrop 1000 UV-Vis spectrophotometer (DeNovix DS-II spectrophotometer, DeNovix Inc., Wilmington, DE 19810, USA). DNA was then diluted to a concentration of 20 ng/µl for PCR assay. The nicosulfuron-resistant johnsongrass from Nebraska used in this study possessed the Trp 574 Leu mutation 39 . Hence, single nucleotide polymorphism (SNP) primers targeting a unique short-range haplotype of Inzen ® sorghum (Val 560 Ile + Trp 574 Leu) were performed using the PCR Allele Competitive Extension (PACE) platform to confirm the resistant plants 43 . The SNP primers and the PACE genotyping master mix were obtained from Integrated DNA Technologies (IDT) Inc. (Coralville, IA) and 3CR Bioscience (Harlow CM20 2BU, United Kingdom), respectively. In addition to the two no-template controls (NTCs), two nicosulfuron-resistant johnsongrass, one wild-type johnsongrass, and one Inzen ® sorghum were also used in the PCR.
The PCR was performed according to the manufacturer's protocol (Bio-Rad Laboratories, Inc., Hercules, CA), with denaturation for 15 min at 94 °C, followed by 10 cycles of denaturation at 94 °C for 20 s, annealing where, y is the mortality (%), x is the herbicide dose (g ai ha −1 ), b is the slope around e, c is the lower limit (theoretical minimum for y normalized to 0%), and e = LD 50 (inflection point, mid-point or estimated herbicide dose when y = 50%). Windrose plots that represented wind speed and frequency during the flowering window in each of the eight directions were created using a macro in Microsoft Excel. Progeny seedling survival (%) that represents gene flow (%) was determined using Eq. (3).
where, X is the number of plants that survived the herbicide application, Y is the total number of plants sprayed for ith distance in jth direction.
To test whether gene flow frequencies varied among the directions, ANOVA was conducted using JMP PRO v.14 (SAS Institute, Cary, NC, USA), based on the average gene flow frequency values in each direction; ENVs were considered as replicates in this analysis. A non-linear regression analysis for gene flow rate, describing an exponential decay function (Eq. 4), was fit using SigmaPlot based on the gene flow frequencies observed at different distances pooled across the directions and ENVs.
where, y is the PMGF (%), x is the distance (m) from pollen source, y0 is the lower asymptote (theoretical minimum for y normalized to 0%), a is the inflection point, mid-point or estimated distance when y = 50%, and b is the slope around a.
A Pearson correlation analysis was conducted to determine potential association between PMGF [overall PMGF, short-distance PMGF (5 m), and long-distance PMGF (50 m)] and the environmental parameters temperature, relative humidity, and dew point. Further, a correlation analysis was also conducted to understand the association between PMGF frequencies and specific wind parameters such as wind frequency, wind speed, and gust speed. The molecular data were analyzed using KlusterCaller 1.1 software (KBioscience).

Results and discussion
Dose response. The dose-response analysis showed that the AS biotype (i.e. susceptible johnsongrass) did not survive the field rate (1×, 63 g ai ha −1 ) of nicosulfuron; the majority of AS plants died at the 0.5 × rate (Fig. 2). However, the AR biotype showed strong resistance to nicosulfuron, with survival up to the 32 × rate, the highest www.nature.com/scientificreports/ rate tested here (Fig. 3). The LD 50 values were 5.6 and 166.3 g ai ha −1 , respectively for the AS and AR biotypes, with an R/S ratio of 30. Based on the dose-response assay, a discriminant dose of 95 g ai ha −1 (1.5×) was used to select hybrid progenies that contained the resistance-conferring mutation. At this dose, the heterozygote hybrid progeny is expected to survive the herbicide, whereas none of the susceptible wild-type progeny would survive.
Werle et al. 44 tested the resistance level of putative ALS-inhibitor resistant johnsongrass biotypes collected from Nebraska and Kansas and reported a 4.9 (J-35) and > 1000-fold resistance (J-36) in two resistant johnsongrass biotypes, which required 0.61 and 446.4 g ai ha −1 nicosulfuron, respectively to achieve 50% growth reduction. The biotype used here had a relatively lower level of resistance than that of J-36, yet was sufficient to effectively discriminate between the AS and AR biotypes. Thus, the AR biotype was an appropriate genotype for utilization in this gene flow study.  Table S1 and Fig. 4). At the 5 m distance, PMGF ranged from 0 to 45% across the directions and ENVs. The average PMGF (averaged across directions within an Env) at the 5 m distance varied from 9.6 to 16.2% across the three ENVs, which progressively declined with the increasing distance from the AR block (Table 1). At 50 m, the farthest distance investigated here, the average PMGF ranged from 0.8 to 1.2% across the ENVs; however, it could be as high as 8.7% in a specific direction in a particular ENV (Supplementary Table S1). The windrose plots describe the frequency and speed of wind events in different directions ( Fig. 4A-C). A wheel diagram demarcated with the gene flow (%) in each direction is presented (Fig. 4D-F), and the entire gene flow dataset for each direction and distance for the three ENVs is provided in the Supplementary Table S1. The exponential decay function developed based on PMGF recorded at each increasing distance from the pollen source across the directions and ENVs is shown in Fig. 5. The model predicted 50% reduction in PMGF at 2.2 m and 90% reduction at 5.8 m from the pollen donor block.
The current study provides a detailed account of hybridization potential between johnsongrass biotypes across multiple ENVs. We used percent hybridization as a measure of PMGF because gene flow should include successful integration of the trait in the progeny 47 . While hybridization between sorghum and johnsongrass was studied earlier 48,49 , to our knowledge this is the first study that investigated PMGF between two johnsongrass biotypes. The study by Arriola and Ellstrand 27 reported an outcrossing frequency of up to 2% in johnsongrass at 100 m distance from sorghum; it is notable that ploidy difference between sorghum (2n = 2X = 20) and johnsongrass  www.nature.com/scientificreports/ (2n = 4X = 40) is expected to reduce outcrossing frequency between the two species 34 . Considerable outcrossing frequencies documented in the current study at 50 m distance is not surprising, especially given that there are no ploidy barriers between johnsongrass biotypes. Though the distance of pollen movement has not been studied in johnsongrass, grass pollen is typically known to travel for long distances, often facilitated by wind dispersal. Switchgrass (Panicum virgatum L.) pollen, for example, can travel as far as 3.5 km 50 . In addition to potential long-distance pollen dispersal, the long and precociously exserted stigma of johnsongrass, combined with its xenogamous breeding system, open panicles, and extended flowering periodicity increases the opportunities for outcrossing 25 . Although reports suggest that PMGF is a natural phenomenon in Sorghum spp., leading to a wide range of hybridization among members of this genus 15,51-53 , there is a dearth of information regarding intraspecific PMGF in johnsongrass. The current study offers valuable insights in this aspect.
Overall, PMGF in johnsongrass is generally high compared to a number of other agronomically important grass weeds. For example in barnyardgrass, 0.01% PMGF was detected at 50 m in Arkansas 37 , whereas in wild oats (Avena fatua), 0.09% PMGF was detected at 56.4 cm distance in Canada 54 . The PMGF frequencies observed between johnsongrass biotypes have significant implications for novel trait movement among Sorghum spp. complex in agricultural landscapes. In particular, PMGF can contribute to the spread of herbicide resistance genes among field populations, with significant agronomic implications 55 .
In addition to the breeding compatibility between pollen donor and recipient plants, the frequency of PMGF also depends on pollen density and size of the recipient population 56 . Pollen density is greatly influenced by the size of the pollen donor block 57 ; large pollen source fields can lead to high levels of gene flow at greater distances [58][59][60] . In the current study, both the pollen donor and recipient populations were at the small-plot   Influence of environmental parameters. The effects of environmental conditions, especially temperature, relative humidity, and dew point, on PMGF were evident in the current study. The ENV1 (summer environment) generally had lower average PMGF values compared to ENV2 and ENV3 (fall environments). PMGF within short distance (i.e. 5 m) was negatively correlated with temperature (r = 0.7, p < 0.05) (Fig. 6), but positively with humidity (r = 0.9, p < 0.001). The temperatures were generally cooler and humidity levels were greater in ENV2 and ENV3, with high levels of corresponding PMGF frequencies. Pollen and stigma, the two indispensable reproductive organs involved in pollination, are highly sensitive to environmental stressors such as hot and dry weather conditions 62,63 , likely explaining the relatively low average PMGF rates in ENV1. At the greater distance of 50 m, PMGF showed a negative correlation with RH and a positive correlation with temperature. High relative humidity typically increases the weight of pollen grains and thereby reduces the distance of pollen transport through wind 64 .
The wind parameters, especially the frequency of wind in a given direction did not show a consistent association with PMGF (Fig. 4). Although PMGF was strongly correlated with distance from the pollen source and pollen was predominantly dispersed by wind, correlation analysis failed to provide a strong association between PMGF and wind parameters (Fig. 7). For example, at the 5 m distance in ENV1, PMGF in the E direction appeared to be consistent with the wind frequency, whereas in the W and NW directions wind flow was infrequent, yet recorded the highest PMGF rates (32.3% and 21.3%, respectively). On the other hand, frequent wind flow was observed in the SE direction, but PMGF was relatively low at 3.6% (Fig. 4A,C). Similar observations were also noted in ENV2 and ENV3.
The frequency, speed, and direction of wind flow were highly variable within and among the ENVs (Fig. 4A-C) and so were the wind gusts (data not shown). The most common wind speed was 0-2 m s −1 followed by 2-4 m s −1 across the ENVs (Fig. 4A-C). Even though wind frequency and speed highly varied across the directions, almost all directions received at least some frequency of wind, which might have moved a considerable amount of pollen, especially at closer distances, but subsequent pollination might have been influenced by several other factors. More importantly, the random wind gusts might have greatly contributed to the lack of association observed here because gust events can move pollen in an unpredictable fashion at different directions and distances. This might explain the high PMGF frequencies observed at 5 m distance in W and NW directions in ENV1 (Fig. 4A,D), though the wind frequency and speed were low.
Sarangi et al. 38 reported a significant association of wind frequency and speed with gene flow (%) in waterhemp (Amaranthus tuberculatus) in Nebraska, as opposed to our findings. However, they used a clean field in their study, where all the plants other than the pollen recipient blocks were killed, which was also the case with Bagavathiannan and Norsworthy 37 while studying PMGF in barnyardgrass (Echinochloa crus-galli) in Arkansas. In the current study, the presence of tall and dense weed vegetation between the pollen donor and recipient blocks might have diffused pollen movement, affecting pollen flow patterns. Thus, PMGF was highly random at greater  Molecular confirmation of hybrids. The hybrids were first confirmed phenotypically based on survival to the discriminant dose of nicosulfuron; the susceptible plants were dead within 14-21 days, whereas the resistant ones survived the application (Fig. 3). Survival to herbicide is a convenient assay for scoring PMGF in this case. However, the molecular investigation conducted on randomly selected plants that survived the herbicide application served as a confirmative test. The Trp 574 Leu mutation, which is present in the AR plants and confers ALS-inhibitor resistance, was detected in the plants determined to be hybrid progenies (Fig. 8). The molecular  www.nature.com/scientificreports/ detection of the Trp 574 Leu mutation was a straight-forward process, and provided definitive confirmation of the hybrid progeny.

Conclusions
Results of this study offer a baseline estimate of PMGF in johnsongrass. Significant levels of PMGF occurred from AR to AS johnsongrass biotypes under the experimental conditions in southeast Texas. PMGF was detected even at a 50 m distance from the resistant pollen source, which was the farthest distance studied here. However, findings suggest that gene flow in this species could occur for much farther distances, though the frequencies are expected to be much lower. Several factors such as the size of pollen-donor and recipient populations, the speed and direction of wind gusts, temperature, and humidity can greatly influence the degree of PMGF under production field conditions. This is the first study of its kind that demonstrates the significance of PMGF for the transfer of herbicide resistance between field populations of johnsongrass. Thus, field management programs should consider eliminating opportunities for PMGF through various agronomic management approaches. In this regard, best management practices should include management of late-season (flowering) johnsongrass as well as field-edge and roadside populations. Future research should include a more elaborate investigation of PMGF in johnsongrass under production-scale field conditions.

Data availability
All data used in the manuscript can be accessed in Dryad Data Repository (will be submitted upon formal acceptance of the manuscript).